Summary: Engagement of multicomponent immunoreceptors such as the T cell antigen receptor results in rapid recruitment and activation of multiple protein tyrosine kinases (PTKs) including Lck, Fyn, ZAP-70 and Itk. These PTKs then phosphorylate many enzymes and adapter molecules involved in complex signaling cascades. Our studies have focused on a critical substrate of the PTKs, LAT (linker for activation of T cells), a 36-38kD integral membrane adapter protein. We have performed studies to characterize how LAT is phosphorylated and then binds many critical signaling molecules, thus bringing other adapter molecules and enzymes in multimolecular complexes to the plasma membrane in the vicinity of the activated TCR. Biochemical, biophysical, microscopic and genetic techniques are currently employed to study the characteristics of LAT-based signaling complexes and the enzyme pathways that are coupled to and activated at LAT complexes. In the past year we have published several studies that can be viewed as a continuation of efforts to bring cutting-edge microscopic techniques to the study of T cell activation. Previously we demonstrated that LAT-based complexes known as microclusters are the site of molecular complex formation occurring with T cell activation. Use of new super-resolution microscopy techniques led to insights in the nanostructure of these complexes in cells. One form of super-resolution microscopy, photo activation localization microscopy (PALM), was used extensively in our studies of signaling complexes. In a 2011 paper we performed two-color microscopy with this technique and demonstrated the nanostructure of complexes in cells. In subsequent work, we developed a novel method of using three photoactivable reagents to enable simultaneous analysis of three different molecules in fixed cells. Last year we used PALM to demonstrate that LAT and another signaling molecule, SLP-76 initially are randomly dispersed in clusters at the onset of T cell activation. The nanostructure that we previously discovered develops over time and requires T cell activation and an intact actin cytoskeleton. We concluded in this publication that the structure and content of LAT-based complexes vary over the course of T cell activation. PALM is one form of a super-resolution technique known generally as single-molecule localization microscopy. Another technique in this category of super-resolution microscopy is stochastic optical reconstruction microscopy (STORM). We adapted this technique to the study of the microclusters formed during T cell activation. We introduced fluorescent nanodiamonds as fiducial markers, which enabled very high localization precision in the STORM images. Additionally, we developed a multiplexing technique that allowed us to study as many as 25 different molecular epitopes within the microclusters. This technique offers the hope of further defining the spatial relationships between various molecules in microclusters. An additional article provided practical advice in performing such studies and another manuscript reviewed super-resolution techniques. Our laboratory previously reported on the development of a conditional knock-out of the Ras activator molecule, SOS1 in T cells. Absence of this molecule in the T cell lineage resulted in decreased thymic cellularity and other defects. Other studies in which this mouse was crossed to mice with other deletions followed. Over time a mouse lacking both SOS1 in T cells and a related enzyme, SOS2, developed an unexpected cartilage phenotype. Joint nodules were the initial observation and joint stiffness, hind limb paralysis and lameness followed. Chondrocyte dysplasia was the underlying cause of these symptoms. This dramatic phenotype could be due to CD4-Cre expression with SOS1 deletion in non-conventional T cells or chondrocytes. As such the results also indicate that investigators should be cautious in attributing results of experiments dependent on CD4-Cre targeting only to gene knock-out in conventional T cells.